Edta free protease inhibitor

THP-1-derived macrophages or BMDMs were seeded in 6-well plates and treated as indicated. The cell pellets were collected into 1.5 ml Eppendorf tubes and lysed in TBS buffer (50 mM Tris-HCl (GCRF, China), 150 mM NaCl (GCRF, China), 0.5% Triton X-100 (Sigma-Aldrich), PH 7.4) with phosphatase inhibitor (Roche) and EDTA-free protease inhibitor (Bimake) on a rocker for 30 min on ice, and then centrifuged at 6000 × g/4 °C for 15 min to discard the supernatants. The cell supernatants were harvested for precipitation and detected the activation of caspase-1 (p10) and the maturation of IL-1β (p17) by western blotting. The pellets were washed twice with TBS buffer and resuspend in TBS buffer containing 2 mM fresh disuccinimidyl suberate (DSS, Thermo Fisher Scientific) cross-linker at 37 °C for 30 min to crosslink with flipping the tubes every 10 min, and then spun at 6000 × g/4 °C for 15 min. The crosslinked pellets were resuspended in 25 μl 2× SDS loading buffer (Cell Signaling Technology) and boiled at 100 °C for 5 min, and analyzed by immunoblotting of anti-ASC antibody, anti-BAX antibody or anti-BAK antibody.

BMDMs were cultured in 6-well plates and exposed to apilimod for a duration of 2 hours. Following this, the cells were lysed using TBS buffer, which consisted of 50 mM Tris-HCl (purchased from GCRF, China), 150 mM NaCl, and 0.5% Triton X-100 (purchased from Sigma-Aldrich), with a pH of 7.4. Additionally, phosphatase inhibitor (purchased from Roche) and EDTA-free protease inhibitor (purchased from Bimake) were added to the lysate. The cells were incubated on a shaker for 30 minutes at 4 °C and then centrifuged at 6000 × g/4 °C for 15 minutes to remove the supernatant. The pellets were washed twice with TBS buffer, and then resuspended in TBS buffer containing 2 mM fresh disuccinimidyl suberate (DSS, purchased from Thermo Fisher Scientific). The cross-linking was carried out at 37 °C for 30 min with the tubes being flipped every 10 minutes. The samples were then centrifuged at 6000 × g/4 °C for 15 minutes. The cross-linked pellets were resuspended in 25 μl of 3 × SDS loading buffer and boiled at 100 °C for 10 min. Finally, the samples were analyzed by immunoblotting using an anti-ASC antibody.